To analyze whether specific protein-lipid interactions or physical features of the membrane contribute to cytochrome P450SCC (CYP11A1) activation by lipids, dimyristoylphosphatidylcholine/cardiolipin and dimyristoylphosphatidylcholine/branched phosphatidylcholine vesicles of defined acyl chain structure were studied for their ability to stimulate the side chain cleavage activity of the enzyme. Activation was found to increase with the mole percent of nonbilayer lipids in the system and the chain lengths of both the branched and main fatty acyl chains of the activator lipid. Unsaturation provided by dioleoylphosphatidylcholine as host lipid leads to a further increase in the potency of the branched phosphatidylcholines to activate the enzyme. The observed activation can be qualitatively interpreted in terms of the effect of these lipids on the hydrophobic volume of the membrane. Using differential scanning calorimetry, we showed that the branched phosphatidylcholines perturb the bilayer membrane structure of dimyristoylphosphatidylcholine and lower the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine, i.e., promote hexagonal phase formation. We also examined the effect of eicosane on both the cytochrome P450SCC activity and the lipid polymorphism and found that eicosane increases both the activity and the hexagonal phase propensity of the vesicle membrane. Because of these correlations, we conclude that the nonbilayer phase propensity of the membrane rather than specific binding of activator lipids to the enzyme explains best the observed activation of enzymatic activity by the lipids.

Phosphatidylcholines (PCs) with branched fatty acyl chains substituted in the two positions of the main chains (branched PCs) have been shown to be potent activators of the side chain cleavage activity of cytochrome P450SCC (CYP11A1) (Schwarz, D., Kisselev, P., Wessel, R., Jueptner, O., and Schmid, R. D. (1996) J. Biol. Chem. 271, 12840 -12846). The present study reports on the effect of a series of branched PC on cholesterol binding, membrane integration, and protein exchange in large unilamellar vesicles prepared by an extrusion technique. Enzyme kinetics using vesicles as well as optical titration using a micelle system with the detergent Tween 20 demonstrate that activation is correlated with the fraction of P450SCC in the high spin form. The potency of branched PCs both to activate the enzyme and to induce spin state changes increases with increasing lengths of both the branched and main fatty acyl chains. We found that the extent as well as the rate of integration of P450SCC into vesicle membranes studied by gel chromatography and stopped flow kinetics were increased by branched PC. Finally, it is demonstrated by measurement of the enzymatic activity in primary and secondary vesicles that branched PCs are potent in retaining a very rapid exchange of P450SCC between vesicles, in contrast to cardiolipin, that partially inhibits this exchange process. The data suggest that different properties of P450SCC in membrane systems including cholesterol binding, membrane integration, and protein exchange are affected by branched PCs and probably by other phospholipids, too, and therefore must be considered in an explanation of the observed high stimulation of activity.Cytochrome P450SCC (CYP11A1) is an integral mitochondrial enzyme that is located on the matrix face of the inner membrane of mitochondria. It catalyzes the side chain cleavage of cholesterol to yield pregnenolone, the common precursor of steroid hormones. Enzymatic studies employing both solution (micelle) and membrane (vesicle) systems demonstrated stimulation of activity to varying extent by phospholipids depending on the phospholipid used, the kind of reconstitution, size of vesicles, and small amounts of impurities (for a review see Ref. and references therein and Refs. 2-4). Under the lipids used in reconstitution CL1 played a special role mainly because (i) it is a typical mitochondrial lipid and (ii) it represents the most potent activator of P450SCC activity (5-7). With regard to the mechanism of activation, CL is of further interest because of the exceptional structure of its hydrophobic part, i.e. consisting of four fatty acyl chains instead of two of most other phospholipids.It has been recently reported by us that certain branched PCs that in the structure of their hydrophobic part are similar to CL but have fully saturated acyl chains cause potent activation of P450SCC comparable only with those by CL (8). By systematic variation of the lengths of both the branched and main chains of these lipids and systematic introduction of unsaturation,...

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